The long term goal of the proposed research is to determine the mechanism of free fatty acid (FFA) transport across adipocyte membranes. Circulating FFA are predominantly derived from adipose tissue. Regulation of FFA is critical because FFA provide a major portion of energy needs and elevated levels adversely affect health. Even relatively modest elevations may play an important role in reducing insulin sensitivity in type II diabetes and significantly increase the risk of sudden death. Much greater increases occur acutely, for example in cardiac ischemia and stroke, and these elevations may adversely affect prognosis by inducing arrhythmias, promoting myocyte apoptosis and increasing the risk of severe hemorrhagic events. These increases in circulating FFA are due to net efflux from adipocytes. Therefore understanding whether the adipocyte plasma membrane plays a role in regulating net influx is critical in human health and disease. There is however no consensus about this issue and the key question is whether FFA transport occurs rapidly through the lipid phase or whether transport is regulated by a membrane carrier protein. During the first 4 years of this grant we have demonstrated that transport across lipid bilayers is rate limited by slow flip-flop and that previous reports of rapid flip-flop are invalid. We have obtained strong evidence for a carrier protein in whole adipocytes and consistent results in plasma membrane vesicles. However, the carrier revealed by our studies has characteristics that are unprecedented, including: 1) an ATP dependent FFA pump, 2) influx/efflux asymmetry, 3) an efflux gate, 4) a plasma membrane whose lipid phase is highly refractory to FFA flip-flop, and 5) saturable transport without a "diffusive" component. In the continuation of this project we will: 1) determine if lipid vesicles can be formed which reproduce the extremely slow FFA transport across the lipid phase of the adipocyte plasma membrane, 2) determine the mechanism of transport across adipocyte plasma membrane vesicles and 3) determine if the transport properties of the whole adipocytes are only consistent with a transport protein. In that event we will begin studies to identify the carrier in adipocytes and their plasma membranes. These studies will be done using RNA interference, multi imaging mass spectroscopy and new forms of ADIFAB the fluorescent probe of FFA to monitor FFA transport. [unreadable] [unreadable]